Abstract
Formate-nitrite transporters (FNTs) facilitate the translocation of monovalent polyatomic anions, such as formate and nitrite, across biological membranes. FNTs are widely distributed among pathogenic bacteria and eukaryotic parasites, but they lack human homologues, making them attractive drug targets. The mechanisms and energetics involved in anion permeation across the FNTs have remained largely unclear. Both, channel and transporter mode of function have been proposed, with strong indication of proton coupling to the permeation process. We combine molecular dynamics simulations, quantum mechanical calculations, and pKa calculations, to compute the energetics of the complete permeation cycle of an FNT. We find that anions as such, are not able to traverse the FNT pore. Instead, anion binding into the pore is energetically coupled to protonation of a centrally located histidine. In turn, the histidine can protonate the permeating anion, thereby enabling its release. Such mechanism can accommodate the functional diversity among the FNTs, as it may facilitate both, export and import of substrates, with or without proton co-transport. The mechanism excludes proton leakage via the Grotthuss mechanism, and it rationalises the selectivity for weak acids.
Highlights
Formate-nitrite transporters constitute an ancient family of transmembrane proteins involved in the translocation of monovalent anions across biological membranes[1,2]
In order to determine the energetics of permeation across the Formate-nitrite transporters (FNTs), we calculated PMFs for permeation of multiple substrates across NirC, HSC, VcFocA, and EcFocA, considering all plausible combinations for the protonation states of the central histidine and the permeating substrate
All tested anions encounter a very high permeation barrier of 60 to 105 kJ mol−1 when the central histidine is in its neutral form (Fig. 2, top row), which is in good agreement with previous calculations involving formate permeation across VcFocA31
Summary
Formate-nitrite transporters constitute an ancient family of transmembrane proteins involved in the translocation of monovalent anions across biological membranes[1,2]. We demonstrate the functional roles of the proton and the central histidine in the permeation process, as well as the importance of the conformation of the Ω-loop in FocA. To this end, we used molecular dynamics (MD) simulations to calculate potentials of mean force (PMFs, sometimes referred to as “free energy profiles”) for permeation across the FNTs, while considering different protonation states of the central histidine and permeating substrate. The simulations suggest a necessity for substrate protonation in order to complete the permeation This mechanism was further investigated in NirC, for which we performed additional sets of free energy calculations and quantum mechanics/molecular mechanics (QM/MM) simulations, revealing a quantitative picture of anion permeation across the FNTs
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